Histochemistry

Histochemistry (1989) 92: 161-169

9 Springer-Verlag 1989

Ultrastructural study of the distribution of calcium in the pineal gland of the rat subjected to manipulation of the photoperiod M.D.L. Pizarro, F.E. Pastor, A. L6pez Gil, and L. Mufioz Barragfin* Department of Investigation and Electron Microscopic, Hospital Clinico, Paseo de S. Vicente, s/n, E-37007 Salamanca, Spain Received December 2, 1988 / Accepted January 26, 1989

Summary. Using the pyroantimoniate technique, a study was conducted at electron microscope level on the distribution of the calcium ion in the pineal glands of normal adult Sprague-Dawley rats with initial weights of 150-200 g subjected to a 12:12 light dark cycle and others under the same conditions were subjected to modifications in the noradrenergic signal, such as continuous illumination over 7 days, blinding by bilateral enucleation (7 or 90 days) before sacrifice and bilateral superior cervical gangliectomy at 21 days before sacrifice. All the animals were sacrificed by decapitation, half of them at midday and the other half at midnight. Abundant fine precipitations of calcium were found in the intercellular spaces of the pineal glands of the normal rats. By contrast, in the gangliectomized animals subjected to constant illumination and chronic binding these precipitations were few in number. Additionally, two types of pinealocytes were observed regarding the distribution and concentration of intracytoplasmic calcium in both the normal and experimentally manipulated animals. Type I correspond to the classic light pinealocytes, with an absence of intracytoplasmic precipitations, although in the normal and gangliectomized animals sacrificed at midnight it was possible to observe fine deposits inside the mitochondrial matrix. Type II correspond to the classic dark pinealocytes, with a dense cytoplasmic matrix and numerous deposits of intracytoplasmic and intranuclear calcium; these were never seen in the type I pinealocytes.

Introduction Study of calcium in the pineal gland has aroused great interest in view of the well known process of progressive calcification undergone by this organ (Del Rio Hortega 1932; Tapp and Huxley 1972; Zimmerman and Bilaniuk 1982). In fact, the presence of calcareous concretions has been reported in many animals including man (Del Rio Hortega 1932; Kitay and Altschule 1954; Scharenberg and Liss 1965; Lukaszyk and Reiter 1975; Japha et al. 1976; Diehl 1978; Allen et al. 1981). The incidence of these formations varies considerably within each animal species and between individuals (Welsh 1985; Trentini et al. 1986) and their appearance cannot be considered as an involutive signal of * To whom offprint requests should be sent

the pineal gland (Tapp and Huxley 1971) but rather should be related to the gland's metabolic activity (Lukaszyk and Reiter 1975). The calcium signal is also an important component in the noradrenergic regulation of the pineal gland (Sugden et al. 1987) and an increase in the intracellular pool of the ion is required for the synthesis of cyclic nucleotides (Sugden et al. 1986). This increase also facilitates translocation of the protein kinase C from the cytoplasm to the membrane, thus favouring the activation of adenyl-cyclase (Klein et al. 1987). Regarding this, different ultrastructural studies have demonstrated the subcellular distribution of calcium in the pineal gland of mammals (Welsh 1984; Krstic 1985, 1986; Pizarro et al. 1988), using potassium pyroantimoniate as the precipitating agent (Appleton and Morris 1979; Eisenmann et al. 1979). In the present work we analyzed the subcellular distribution of pineal calcium in normal rats and in rats subjected to experimental states aimed at modifying the noradrenergic signal that induces monoamine synthesis by the pinealocytes.

Materials and methods Albino Sprague-Dawley rats with a weight range of 150-200 g subjected since birth to a 12:12 light/dark cycle (lights on at 06.00 a.m) were used; these were divided into the following groups of 8 animals each: a) Normal rats that had not previously undergone any experimental protocol ; b) Animals that had undergone denervation of the pineal gland by bilateral superior cervical gangliectomy 21 days prior to sacrifice. Denervation of the gland was carried out under anaesthesia with sodium thiopental (30 rag/100 g, i.p.); c) Animals subjected to continuous illumination over 7 consecutive days, and d) Animals blinded by bilateral enucleation at 7 and 90 days before sacrifice. Half of the animals were sacrificed by decapitation at midday, removing the gland under daylight, and the other half at midnight. In the latter case, the pineal glands were removed under red light. Immediately after removal the pineal glands were fixed in a 3% solution of glutaraldehyde containing 2% potassium pyroantimoniate over 45 min at 4~ C, according to the method of Leuschen et al. (1981). Following the protocol of this authors, post-fixing was done in a 1% solution of osmium tetroxide in distilled water containing 2% potasium pyroantimoniate over 1 h at 4~ C. The pH of both solutions was adjusted to 7.3 with acetic acid (0.01 N) and KOH (0.01 N). After fixing and post-fixing, the tissue was thrice-washed in a 7% solution of sucrose for 10 rain each time.

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Fig. 2. Electron micrograph of pineal gland of a normal rat treated with potassium pyroantimonate. Note two type I pinealocytes (LP) and one type II pineatocyte (DP). Abundant precipitates are dis,11

Fig. 1. Electron micrograph of pineal gland of a normal rat treated with potassium pyroantimonate. Abundant precipitates of calcium are seen in the intercellular spaces located between many light pinealocytes (type I pinealocytes). L P = L i g h t pinealocytes; Ly= lisosomes. • 45500

persed throughout the cytoplasm of the DP. Note accumulation of precipitates in the nucleus of this cell (arrows) that does not affect the nucleolus (NL). x 21900 After the final wash, the pieces were dehydrated and embedded in araldite for study under a conventional electron microscope. Half of the ultrathin sections from each gland were studied without contrasting; the other half were contrasted with the Reynolds (1963) method. The pieces were studied under a Philips EM-201 electron microscope.

Fig. 3. Transversal section of the cytoplasmic process of a type I pinealocyte, showing five mitochondria, whose matrix contains fine precipitates of calcium. Note absence of intracytoplasmic precipitates, which are abundant in the intercellular space, x 45 500 Fig. 4. Ultrathin section of pinealocytes of rats treated with potassium pyroantimonate. Note presence of fine precipitates of calcium in the intercellular space and absence of these in the vesicles of the synaptic ribbons, x 45 500 Fig. 5. Section of pineal gland of rat treated with potassium pyroantimonate. Note presence of fine prolongations of type II pinealocyte, full of precipitates of calcium, (arrows) surrounding a type I pinealocyte. N: nucleus; G: Golgi complex; L: lipoid droplets, x 11200 Fig. 6. Ultrathin section of a cytoplasmic process of a type II pinealocyte showing numerous microtubules and precipitates of calcium. • 69 500

Fig. 7. Electron micrograph of pineal gland of rat treated with potassium pyroantimonate showing several type t pinealocyte (LP) and an interstitial cell (IC). Note abundance of calcium precipitates in all the intercellular spaces and absence of these in the cytoplasm of both the interstitial cell (IC) and the LP. x 21900

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167 Results

1. Calcium distribution in the pineal gland of normal rats Intercellular space and pinealocytes. Abundant fine precipitations of calcium were observed in the intercellular spaces of the pineal glands of the normal rats both when sacrificed at midday and at midnight (Fig. 1). Two types of pinealocytes were distinguished (Fig. 2) regarding the distribution and concentration of intracytoplasmic calcium. Type I corresponded to the classic clear pinealocytes, with an almost complete absence of intracytoplasmic precipitations, although fine deposits of calcium were observed in the mitochondrial matrix. These latter were particularly striking in the normal rats sacrificed at midnight (Fig. 3). No Ca 2 + deposits were ever observed inside or surrounding the vesicles of the synaptic ribbons or field ribbons and the former, when present, were not particularly outstanding in the intercellular spaces located in the vicinity of the synaptic and field ribbons (Fig. 4). Type II corresponded to the classic dark pinealocytes, with a dense cytoplasmic matrix, m which it was consistently possible to demonstrate very abundant deposits o f C a 2 + inside the cytoplasm, irregularly distributed among the mitochondria, the R E R and other cytoplasmic organelles. These deposits were also seen on the cytoplasmic processes surrounding the type I pinealocytes (Fig. 5) or arranged among the parallel sheaths of microtubules observable in longitudinal sections of the processes (Fig. 6). By contrast, Ca ~ + was only occasionally observed in the mitochondriat matrix of these pinealocytes, both in the rats sacrificed at midday and at midnight. A consistent finding in the type II pinealocytes was the presence of intranuclear deposits of calcium; these were never in the type I cell. Both cell types were seen in the pineal gland of all the normal rats studied, regardless of the time of their sacrifice.

Interstitial cells. No intracytoplasmic or intranuclear calcium deposits were seen in macroglial cells (oligodendrocytes) (Fig. 7), microglial cells rich in lysosomes, although fine precipitations were detected in the intercellular spaces between the pinealocyte membrane and the interstitial cells.

4

Fig. 8. Ultrathin section of pineal gland of rat treated with potassium pyroantimonate. Note presence of fine sympathetic nerve fibres and abundant precipitates of calcium (arrows) in the intercellular spaces between the former and the type I pinealocyte (LP). Note the absence of basal lamina between the nerve fibre and the plasma membrane of the pinealocyte, x J[03300 Fig. 9. Same images as previous one showing several sympathetic fibres and an LP (upper part of image), separated by the basal membrane (BM). (arrow) Note absence of calcium deposits. x 45500 Fig. 10. Ultrathin section of pineal gland of gangliectomized rat. Note presence of very abundant calcium precipitates in the intercellular spaces and in the anitocbondrial matrix (m) of an LP. x 103 300 Fig. 11. Ultrathin section of pineal gland of rat blinded for 90 days showing portions of four type I pinealocytes. Note the almost complete absence of calcium deposits in intercellular space. Arrows: show isolated precipitates of the cation, x 45 500

Sympathetic fibres and terminals. Although abundant precipitations of Ca z + were observed in the intercellular space between the cell membrane of the pinealocytes and the sympathetic terminals containing granular vesicles with dense cores (Fig. 8), no Ca 2 + was detected around the membrane of the sympathetic fibres separated by the basal lamina of the cell membrane of the pinealocytes (Fig. 9). No calcium was detected inside the sympathetic terminals. 2. Distribution of calcium in the pineal glands of the experimentally-treated rats Types ofpinealocytes. The two types of pinealocytes found in the normal rats were found in all the pineal glands of the different groups of experimentally-treated animals, and the pattern of distribution was also similar. This similarity in distribution was extensive to the C a 2+ present in the mitochondrial matrix, with the exception of the rats sacrificed at 21 days after pineal denervation by bilateral superior cervical gangliectomy sacrificed at midnight, In the latter animals abundant deposits of intramitochondrial calcium were detected that were particularly striking in the mitochondria of the type I pinealocytes (Fig. 10). Intercellular space. In the animals sacrificed at 21 days after bilateral superior cervical gangliectomy or 7 days after being subjected to continuous illumination large areas of the pineal gland were found in which there were scanty precipitations of calcium in the intercellular space. Likewise, in the blinded animals sacrificed to 90 days after bilateral enucleation there was a practically complete absence of calcium precipitations in the intercellular spaces (Fig. 11), regardless of the moment of the photoperiod at which the animals were sacrificed. This was not observed in the rats blinded for only 7 days.

Interstitial cells. The pattern of distribution of calcium in the interstitial cells of the pineal glands of the experimentally - treated animals did not vary significantly with respect to the situation observed in the normal animals. Sympathetic terminals. Regarding the sympathetic terminals, no important differences were detected in the pattern of calcium distribution compared with normal animals, since calcium deposits were only observed in the spaces between the cell membrane of the pinealocytes and the sympathetic terminal when there was no basal lamina in between. Thus, excepting the gangliectomized animals, in which degeneration affecting the sympathetic terminals hinders the study of calcium distribution in them, the cell membrane of the sympathetic terminals isolated from the pinealocytes by the basal lamina was seen to be devoid of calcium precipitations. Discussion

At the level of the pineal gland, the calcium signal is known to be an important component in the noradrenergic regulation of monoamine synthesis mediated by the activation of B-adrenergic receptors that requires an increase in intracellular Ca 2 § to potentiate the synthesis of cyclic nucleotides (Sugden et al. 1986). Furthermore, is its possible that ~-adrenergic stimulation of the intracellular concentration of calcium in the pinealocyte may be related to the some-

168 what enigmatic phenomenon of pineal calcification (Diehl 1978; Sugden et al. 1987). Despite the physiological importance that can be attributed to the calcium ion in the mechanisms of monoamine synthesis of the pineal gland, there are few studies on its subcellular distribution in the pineal gland of the rat (Pizarro et al. 1988), since work has mainly centred in the gerbil (Welsh 1984; Krstic 1985, 1986) an animal whose pineal gland exhibits a degree of calcification similar to that observed in humans (Welsh 1985), whereas in the rat the process is hardly detectable (Erding 1977; Diehl 1978). The choice of the rat as an experimental animal in the present work was based on the desire to obtain information of the pattern of calcium distribution in the pineal gland at different moments of the photoperiod, under situations of assumed over stimulation by blinding (Satodate et al. 1973; Pohl and Gibbs 1978), and after deprivation of stimuli by bilateral superior cervical gangliectomy (Weiss and Costa 1967; Moore 1975) or continuous illumination (Klein and Weller 1972; Upson et al. 1976). To do so, we used the potassium pyroantimoniate technique (Appleton and Morris 1979; Leuschen et al. 1981 ; Wick and Hepler 1982), which has proved to be reliable for ultracytochemical d~monstration of exchangeably bound calcium (Wick and Hepler 1982). In our study we observed a typical pattern of subcellular calcium distribution; the deposits of the ion were always detectable in the intercellular space, with no important morphological differences between the animals sacrificed at midday or at midnight. Regarding intracellular calcium, clear differences can be seen in its distribution, allowing us to classify the pinealocytes into two groups. Type I corresponds to the light pinealocytes described by Arstila (1967), Karasek (1971) and Gusek (1976) in the rat and these cell do not contain intracytoplasmic deposits of calcium that can be appreciated with the pyroantimoniate technique, while the type II pinealocytes correspond to the dark pinealocytes reported by the above authors; these contain abundant intracytoplasmic and intranuclear deposits of Ca 2+. Both types of pineatocyte are present in the pineal gland of the rat under all the experimental conditions investigated in this work. The division of pinealocytes into the light and dark types has been reported in different animals species (Arstila and Hopsu 1964; Gusek et al. 1965; Sheridan and Reiter 1968; Upson et al. 1976; see Pevet 1977, 1981, for review). There are fairly sound reasons for suspecting whether one is dealing with a single cell type at different functional states (Karasek 1983) or whether the differentiation is due to the employement of different systems to fix the pieces (Welsh and Reiter 1978). Some authors have even suggested that interstitial cells could also be included within the dark pinealocytes (Pevet 1977). In the present study it is clear that the type II pinealocytes have synaptic ribbons, dense bodies, lipid droplets and lamellae anulae. These findings confer a secretory nature to the cells and differentiate them from the interstitial cells, which appear surrounded by fine deposits of calcium, observable in the intercellular space separating them from the type I and type II pinealocytes. There are no intracytoplasmic calcium deposits in the interstitial cells. In the search for an explanation for the presence of intracytoplasmic deposits of Ca z + in the type II pinealocytes, one should take into account the possibility of a fail-

ure in the calcium-dependent ATPase-mediated mechanism at the level of the plasma membrane for secreting calcium from the inside of the pinealocyte to the interstitial space, as has been pointed out by Krstic (1985). This failure would also involve intracellular membrane systems such as the endoplasmic reticulnm (Waltz 1982) and above all the mitochondria (Somlyo 1984) owing to their capacity for sequestrating intracytoplasmic calcium. According to Krstic (1985), the appearance of deposits of Ca 2 § in the mitochondrial matrix would be related to degenerative processes occurring in the pinealocytes. This notion seem to be supported by the presence of intramitochondrial accumulations of Ca 2§ observed by us in the pinealocytes of gangliectomized rats, since a failure in noradrenergic information leads to a deficit in the synthesis of monoamines by the pineal gland (Moore 1975). However, in this work we have also obtained morphological data that conflict with such a simple explanation as the one put forwards above. Thus, although intramitochondrial deposits of Ca 2§ were observed by us in both types of pinealocyte, they are particularly striking in the type I pinealocytes of normal rats sacrificed at midnight, the time when melatonin synthesis is most pronounced (Klein 1979). In our opinion, the presence of Ca 2+ within the mitochondria of the type I pinealocytes of rats points to a very active role of these organelles in the turnover of the cation from cytoplasmic matrix towards the mitochondria and viceversa. In this sense, the loss by the mitochondria of the capacity for participating in the intracellular turnover of calcium would account for the scarce presence of this cation in the mitochondria of the type II pinealocytes, thus contributing - together with failures on the part of the cell m e m b r a n e - , to the appearance of the large accumulations of Ca 2 § in the cytoplasmic matrix of such pinealocytes. The fact that the abundant presence of intracytoplasmic Ca 2 § may or may not justify use of the term degenerating cells is still hypothetical, although in favour of such a notion it should be noted that this phenomenon is accompanied by important accumulations of the cation inside the nucleus, as was initially reported by Krstic (1985) in the gerbil. However, on evaluating our results, it should be born in mind that the functional activity of the pineal gland does not seem to be reduced to the synthesis of monoamines, since different peptides have been shown to be present in the gland, such as AVT (Milcu et al. 1963; Buijs and Pevet 1980), T R H (Brammer et al. 1979), L H R H (Joseph 1976), A C T H (Fernstrom et al. 1980), whose morphological substrates have still not been elucidated. The importance of calcium in the regulation of the secretion of peptide hormones (Moriarty 1978) - high lighted by Lukaszyk and Reiter (1975) in the pineal gland - may indicate to a possible relationship between the type II pinealocyte and peptide synthesis. However, the cytoplasmic dense bodies seen in this kind of cell correspond to those observed in type I and in no case has it been possible to observe the presence of calcium deposits in contact with the dense bodies in either type of pinealocyte, as has been demonstrated in peptide secretory bodies in other kinds of endocrine cells (Leuschen et al. 1981). References

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